Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression
ABSTRACT Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.
- SourceAvailable from: Dongli Guan
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- "This is likely due to the absence of Cys1 which could potentially form a disulfide bond with Cys þ 1 and inhibit the intein reaction (Chen et al., 2012). Since many enzymatic reactions can be inhibited by divalent ions such as Zn 2þ (Costello et al., 1997; Larsen and Auld, 1989; Perry et al., 1997), and Zn 2þ was found to inhibit the first N–S acyl shift reaction of SspDnaE, a close homolog of NpuDnaE (Nichols et al., 2003), we tested the ability of Zn 2þ to inhibit the C-terminal cleavage reaction of our engineered NpuDnaE construct. ZnCl 2 (0.5 mM) can effectively inhibit the C-terminal cleavage reaction under non-reducing conditions but has little inhibitory effect in the presence of DTT (Fig. 3). "
ABSTRACT: Rapid and efficient tag removal remains a significant problem in recombinant protein purification. Using an engineered DnaE intein from Nostoc punctiforme, we developed a split intein mediated ultra-rapid purification (SIRP) method for the purification of tagless recombinant protein from E. coli lysate in less than 1 hour. This system exhibits extraordinarily rapid thio-induced C-terminal cleavage with about 50% completion within 30 seconds at both 22 °C and 6 °C. This is the fastest C-terminal cleavage activity reported to date for inteins. Although the reaction kinetics slow down after the first minute, >90% cleavage completion is achieved within 30 minutes at 22 °C, or within 3 h at 6 °C. The ultra-rapid cleavage kinetics are made possible by the positioning of the purification tag at the split junction to the C-terminus of the intein N-fragment, thus avoiding potential steric hindrance of the critical interaction between the N- and C-extein. Target proteins are cleaved to >72% completion after 1 h of intein reaction regardless of the identity of the N-terminal amino acid except in the cases of threonine (50% cleavage) and proline. The C-terminal cleavage reaction can be effectively inhibited by divalent Zn(2+) under non-reducing conditions. Importantly, the association of the intein N- and C-fragments is reversible, enabling the column-bound intein N-fragment bait protein to be regenerated for multiple usages and further reducing the cost of protein purification. SIRP technology should provide a useful tool for the purification of tagless proteins and peptides. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.Biotechnology and Bioengineering 09/2013; 110(9). DOI:10.1002/bit.24913 · 4.13 Impact Factor
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- "Intein T (°C) k (s À1 ) t 1=2 Reference Npu DnaE 37 ð1:1 AE 0:2Þ Â 10 À2 63 s This work Ssp DnaE 23 ð6:6 AE 1:3Þ Â 10 À5 175 min a  Ssp DnaE 23 ð3:3 AE 0:4Þ Â 10 À4 35 min a  Npu DnaE N + Ssp DnaE C 21 ð2:8 AE 0:3Þ Â 10 À5 413 min a  Sce VMA 25 ð2:0 AE 0:3Þ Â 10 À3 6 min a  Ssp DnaB 25 ð9:9 AE 0:8Þ Â 10 À4 12 min a  Mtu RecA 30 n.r. 60–120 min  "
ABSTRACT: We have studied the naturally split alpha subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of (1.1+/-0.2)x10(-2) s(-1) at 37 degrees C. This represents the highest rate reported for the protein trans-splicing reaction so far (t(1/2) of approximately 60s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37 degrees C, and the presence of up to 6 M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.FEBS letters 04/2009; 583(5):909-14. DOI:10.1016/j.febslet.2009.02.003 · 3.17 Impact Factor
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- "The Hedgehog protein is generated as a 45-kDa precursor that undergoes autocatalytic processing to generate an active 22-kDa Nterminal protein (Ho and Scott, 2002). It has been reported that Zinc ions can influence autocatalytic properties of inteins, which exhibit similar autocatalytic processing as Hedgehog (Ghosh et al., 2001; Nichols et al., 2003; Paulus, 2000). However, we failed to detect any changes in the zinc ion concentration following Foi overexpression. "
ABSTRACT: Development of complex organs depends on intensive cell-cell interactions, which help coordinate movements of many cell types. In a genetic screen aimed to identify genes controlling midline glia migration in the Drosophila nervous system, we have identified mutations in the gene kastchen. Here we show that during embryogenesis kastchen is also required for the normal migration of longitudinal and peripheral glial cells. During larval development, kastchen non-cell autonomously affects the migration of the subretinal glia into the eye disc. During embryonic development, kastchen not only affects glial cell migration but also controls the migration of muscle cells toward their attachment sites. In all cases, kastchen apparently functions in terminating or restricting cell migration. We identified the molecular nature of the gene by performing transgenic rescue experiments and by sequence analysis of mutant alleles. Kastchen corresponds to the recently described gene fear-of-intimacy (foi) that was identified in screen for genes affecting germ cell migration, suggesting that Foi-Kastchen is more generally involved in regulating cell migration. It encodes a member of an eight-transmembrane domain protein family of putative Zinc transporters or proteases. We determined the topology of the Foi protein by using antisera against luminal and intracellular domains of the protein and provide evidence that it does not act as a Zinc transporter. Genetic evidence suggests that one of the functions of foi may be associated with hedgehog signaling.Developmental Biology 12/2004; 275(1):245-57. DOI:10.1016/j.ydbio.2004.07.039 · 3.55 Impact Factor